Pericerebral cell targeting extracellular vesicle and drug

Abstract

The invention discloses an extracellular vesicle targeting pericerebral cells and a medicine, and relates to the technical field of biological medicines. According to the extracellular vesicles targeting the pericerebral cells, the extracellular vesicles can be delivered to the pericerebral cells in a targeted mode through the targeting ligand modified on the surface of the extracellular vesicles and the cell surface receptor capable of being specifically combined with the pericerebral cells, and a new delivery tool and therapeutic drug selection are provided for treatment of central nervous system related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an extracellular vesicle and drug that targets brain pericytes. Background Technology

[0002] Pericyte dysfunction is a core pathophysiological mechanism in many central nervous system (CNS) diseases. As specialized vascular wall cells, pericytes densely cover the arteriovenous transition zone and surround the endothelial cells of capillaries (>80%). They interact bidirectionally with endothelial cells, astrocytes, and neurons through direct physical contact and paracrine signals, playing a dominant role in regulating two core functions of the neurovascular unit: dynamically regulating cerebral microcirculation and maintaining the integrity of the blood-brain barrier (BBB). Recent evidence suggests that pericyte loss or dysfunction is involved in the early pathological processes of various CNS diseases, including ischemic stroke, vascular cognitive impairment (VCI), Alzheimer's disease, multiple sclerosis, and optic neuropathy. The mechanism of action involves pericyte damage leading to microcirculatory disturbances and BBB disruption, causing local hypoxia, cellular oxidative stress, and neuroinflammation, triggering and accelerating neurodegeneration and demyelinating lesions. This suggests that pericytes have the potential to serve as an early therapeutic target.

[0003] However, targeted regulation of pericytes faces two common challenges: the efficiency of drug delivery across the blood-brain barrier (BBB) ​​and the specificity of action on pericytes. Tight junctions between brain capillary endothelial cells can block over 98% of small molecule drugs, allowing only lipid-soluble small molecules (<500 Da) or drugs using active transport systems to pass through. Recent studies have highlighted the advantages of extracellular vesicles (EVs), particularly those with a particle size <200 nm, as ideal CNS delivery carriers, including natural biocompatibility, in vivo circulation stability, and BBB penetration. EVs possess a lipid bilayer structure and abundant membrane proteins, encapsulating soluble proteins and nucleic acids derived from their parent cells. After crossing endothelial cells through fusion with the plasma membrane or receptor-mediated endocytosis, EVs can be taken up by pericytes and other central nervous system cells. However, like most central nervous system biologics, natural EVs lack cell targeting specificity. Surface functionalization strategies to achieve cell-specific targeting can further enhance the therapeutic potential of EVs, opening new therapeutic pathways for targeted regulation of pericytes.

[0004] Currently, targeted modification methods for EVs mainly fall into two categories: active intervention in parental cells and passive modification of purified EVs. The former primarily involves fusing the gene sequence of the target protein or peptide with the gene sequence of a selected EV membrane protein through genetic engineering, allowing the parental cells to actively assemble the target protein during EV biogenesis. The latter involves chemically modifying the EVs themselves to bind exogenous substances. Genetic modification is limited to motifs encoded by the target gene, carries a high risk of gene transfer failure and is costly. It is ineffective on pre-isolated EVs and certain difficult-to-transfect cells, and ethical issues such as potential gene transfer risks limit its clinical translation. Compared to genetic modification, chemical modification is simpler, faster, and more flexible. Chemical modification includes both covalent coupling reactions and non-covalent modifications. The former is based on click chemistry and enzymatic reactions occurring on the surface groups of natural EVs, while the latter includes multivalent electrostatic interactions and hydrophobic interactions. Among these, click chemistry, particularly strain-promoted azide-alkynecycloaddition (SPAAC), has become a robust modification strategy. Summary of the Invention

[0005] This invention provides extracellular vesicles and drugs that target brain pericytes. These drugs can be used to directly treat related central nervous system diseases, or as delivery tools to deliver therapeutic drugs to brain pericytes for the treatment of central nervous system diseases.

[0006] This invention is implemented as follows:

[0007] Currently, there is a lack of targeted biologics for the pericytes of the brain. Although therapeutic EVs derived from stem cells, dendritic cells, or macrophages have the ability to cross the brain border (BBB), natural EVs lack cell specificity and are easily taken up non-selectively by other non-target cells in the CNS, affecting their targeted delivery efficiency.

[0008] Surface functionalization is an effective way to endow EVs with cell targeting capabilities. Although genetic engineering methods can achieve stable expression of targeting ligands on EV membranes, these methods suffer from complex production processes, high costs, and potential risks of horizontal gene transfer, limiting their clinical translation prospects. In contrast, post-isolation chemical modification techniques for EVs offer a more clinically feasible alternative.

[0009] Although surface modification technologies, represented by SPAAC click chemistry, have been successfully applied in the engineering of EVs, they still face two major technical bottlenecks in achieving targeted brain cells: first, it is necessary to identify the specific receptors for brain cells and their corresponding high-affinity ligands; second, it is necessary to establish an efficient and stable system for chemical modification of EVs and ligand coupling reaction.

[0010] Based on this, on the one hand, the present invention provides an extracellular vesicle that targets brain pericytes, comprising extracellular vesicles and a targeting ligand modified on the surface of the extracellular vesicles, wherein the targeting ligand specifically binds to cell surface receptors of brain pericytes.

[0011] Optionally, in some embodiments, the targeting ligand is selected from the cyclic peptide cNGR; and the cell surface receptor of the pericytes is selected from CD13.

[0012] Optionally, in some embodiments, the linear precursor amino acid sequence of the cyclic peptide cNGR is: KCNGRC, and its chemical structure is shown in [reference needed]. Figure 1 .

[0013] The cyclic peptide cNGR can specifically bind to CD13, enabling the extracellular vesicles to be specifically targeted to pericytes in the brain.

[0014] Based on the specificity of cNGR's targeted binding to CD13, this invention discloses for the first time the use of cNGR-modified extracellular vesicles to target and bind to pericytes in the brain, thereby achieving the treatment of various CNS diseases involving pericyte loss or dysfunction, as well as drug delivery.

[0015] Optionally, in some embodiments, the targeting ligand is covalently coupled to the surface of the extracellular vesicles.

[0016] Optionally, in some embodiments, the covalent reaction is selected from strain-promoted azido-yne cycloaddition reactions.

[0017] Optionally, in some embodiments, the targeting ligand is coupled to the extracellular vesicle via a DBCO-NHS group.

[0018] The NHS group in the DBCO-NHS ester undergoes a nucleophilic reaction with the primary amino groups (such as lysine residue side chains) of proteins on the surface of EVs, forming a stable amide bond, thereby covalently anchoring the DBCO group to the EV membrane. Subsequently, through the copper-free click chemistry SPAAC, the DBCO group attached to the EVs binds efficiently and specifically to the azide group on the azide-modified cNGR peptide, generating a stable triazole ring linker.

[0019] It should be noted that in other embodiments, the targeting ligand can also be coupled to the surface of extracellular vesicles through some common coupling methods in the art, such as maleimide, biotin-avidin system, etc.

[0020] Optionally, in some embodiments, the extracellular vesicles are loaded with therapeutic or diagnostic agents.

[0021] Optionally, in some embodiments, the therapeutic agent is selected from small molecule chemical drugs such as paclitaxel and curcumin, nucleic acid drugs such as small interfering RNA, and cytokines such as interleukin-12. It should be noted that those skilled in the art can select the appropriate therapeutic agent based on the disease they wish to treat, which is readily achievable for them.

[0022] Optionally, in some embodiments, the extracellular vesicles are extracted from the culture supernatant of human umbilical cord mesenchymal stem cells.

[0023] It should be noted that the extraction of extracellular vesicles is a conventional technique in this field, and those skilled in the art can easily obtain and prepare extracellular vesicles based on existing techniques according to the disclosure of this invention.

[0024] On the other hand, the present invention provides the use of the extracellular vesicles described in any of the above claims in the preparation of drugs for treating central nervous system diseases.

[0025] The results of the embodiments of the present invention show that the aforementioned extracellular vesicles can target pericytes of the brain and exert a direct therapeutic effect, and can be used to prepare drugs related to central nervous system diseases.

[0026] Optionally, in some embodiments, the central nervous system disease is selected from ischemic stroke, VCI, Alzheimer's disease, multiple sclerosis, and optic neuropathy, etc.

[0027] Optionally, in some embodiments, the central nervous system disease is selected from cerebral small vessel disease.

[0028] On the other hand, the present invention provides a composition targeting brain pericytes, comprising extracellular vesicles as described above.

[0029] On the other hand, the present invention provides a medicament for treating CNS diseases, comprising extracellular vesicles as described above, or a composition as described above.

[0030] Optionally, in some embodiments, the central nervous system disease is selected from cerebral small vessel disease.

[0031] Cerebral small vessel disease (CSV) is the leading cause of cerebral microvascular infection (VCI), characterized by white matter damage caused by chronic hypoperfusion (illustrated as high signal intensity in the white matter), ultimately leading to cognitive decline. In this invention, a mouse model of bilateral carotid artery stenosis (BCAS) was used to simulate the pathological process of chronic ischemia and white matter demyelination caused by human CSV. Experimental results showed that the aforementioned extracellular vesicle drug provided by this invention significantly improved white matter demyelination in the model mice. Its mechanism and the drug's ability to specifically target pericytes, through the dual effects of simultaneously enhancing local cerebral microcirculation blood flow and stabilizing the white matter boundary (BBB), fundamentally improved the ischemic microenvironment of the white matter, creating the necessary conditions for protecting and repairing the myelin sheath.

[0032] On the other hand, the present invention provides a method for preparing extracellular vesicles, wherein a targeting ligand is co-incubated with extracellular vesicles to bind the targeting ligand to the surface of the extracellular vesicles, and the targeting ligand specifically binds to receptors on the surface of brain pericytes.

[0033] Optionally, in some embodiments, the targeting ligand is selected from the cyclic peptide cNGR; and the pericyte cell surface receptor is selected from CD13.

[0034] Optionally, in some embodiments, the targeting ligand is covalently coupled to the surface of the extracellular vesicle via linker groups such as DBCO-NHS, maleimide, and biotin-avidin.

[0035] Optionally, in some embodiments, the method includes:

[0036] DBCO-NHS modification steps: 100 μg EVs were reacted with 100 μM DBCO-NHS (532.50 g / mol) at room temperature for 3 hours to obtain DBCO-NHS modified EVs.

[0037] Optionally, in some embodiments, the method includes: a purification step: purifying the product obtained from the DBCO-NHS modification step by centrifuging at 120,000 g and 4°C for 90 min.

[0038] This purification method replaces the 100 kD ultrafiltration tube used in the prior art, which not only shortens the reaction time by 1 hour, but also avoids the use of ultrafiltration tubes, saving material costs. Attached Figure Description

[0039] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0040] Figure 1 The structure of the cNGR polypeptide [(N3-Acp)-KCNGRC(SS)] is given.

[0041] Figure 2 The synthesis steps for cNGR-sEVs.

[0042] Figure 3 The electron microscope shows the morphology of EVs before and after surface functionalization. Surface functionalization does not affect their vesicle shape.

[0043] Figure 4 The nanoparticle tracer analysis showed the particle size of EVs before and after surface functionalization. Surface functionalization slightly increased the particle size of EVs.

[0044] Figure 5 Western blot analysis was performed on the proteins of EVs and human umbilical cord mesenchymal stem cells before and after surface functionalization. Both EVs before and after surface functionalization expressed the marker proteins Alix, CD63, and TSG101, but did not express intracellular α-tubulin.

[0045] Figure 6 Nanoflow cytometry shows that the coupling efficiency of cNGR (with FITC fluorescent group) on the surface of EVs is over 90%.

[0046] Figure 7 Results of intracerebral distribution detection of EVs and cNGR-EVs: A. In vivo imaging showed the intracerebral distribution of fluorescence 2 hours after intranasal administration of EVs and cNGR-EVs. Fluorescence intensity analysis relative to the sham group showed that surface functionalization did not affect the efficiency of EVs entering the brain via the nose. B. Immunofluorescence showed that both EVs and cNGR-EVs could be taken up by pericytes of the corpus callosum (PDGFR-β labeled). Quantitative analysis showed that surface functionalization significantly increased the uptake of EVs by pericytes.

[0047] Figure 8 Results of cNGR-EVs brain distribution detection: A. Laser speckle contrast imaging showed that, compared with the model control group, the blood perfusion in the brain of mice in the cNGR-EVs group was significantly increased; B. Evans blue staining showed that the blue exudate area in the cerebral cortex of mice in the cNGR-EVs group was reduced; C. Transmission electron microscopy images of the corpus callosum region showed that the integrity of myelin sheath structure was improved and demyelinating lesions were alleviated in the cNGR-EVs group. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0049] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0050] Example 1

[0051] extracellular vesicle preparation

[0052] Natural EVs were isolated and extracted from the supernatant of human umbilical cord mesenchymal stem cell culture. Specifically, ultracentrifugation was used: sequential centrifugation at 400 g, 4℃, 5 min; 3000 g, 4℃, 15 min; and 10000 g, 4℃, 30 min was performed to remove cell debris and impurities; finally, the EV pellet was collected by centrifugation at 120000 g, 4℃ for 90 min and resuspended in PBS.

[0053] The obtained EVs were systematically characterized: their typical vesicle morphology was observed by transmission electron microscopy, their particle size distribution and concentration were determined by nanoparticle tracer analysis, the total protein of EVs was quantified by BCA method, and EV marker proteins were detected by Western blot.

[0054] Example 2

[0055] Surface functionalization modification

[0056] DBCO-NHS modification: 100 μg EVs were reacted with 100 μM DBCO-NHS (C25H21N2NaO8S, 532.50 g / mol) at room temperature for 3 hours, and then purified by centrifugation at 120,000 g and 4℃ for 90 minutes to obtain DBCO-NHS modified extracellular vesicles (DBCO-EVs).

[0057] Covalent coupling: The reaction concentration of the azide-coated cNGR peptide [(N3-Acp)-KCNGRC(SS), 816.75 g / mol] was optimized to 10 μM and reacted with DBCO-EVs overnight (8-12 hours) at 4 °C. The free peptide was then removed by high-speed centrifugation to obtain extracellular vesicles covalently coupled with the cNGR peptide (cNGR-EVs).

[0058] Electron microscopy, NTA, and Western blot analysis confirmed that the morphology, particle size, and marker protein expression of EVs remained stable before and after modification. Nanoflow cytometry confirmed that the coupling efficiency of cNGR ligand on the EV surface was over 90%. Figures 3-6 )

[0059] Example 3

[0060] Fluorescent labeling and in vivo tracing

[0061] Fluorescent labeling: 100 μg of EVs were reacted with 100 μM DBCO-NHS (C25H21N2NaO8S, 532.50 g / mol) at room temperature for 3 hours, followed by purification by centrifugation at 120,000 g and 4°C for 90 minutes to obtain DBCO-NHS-modified extracellular vesicles (DBCO-EVs). 10 μM Cyanine3 azide (Cy3) or Cyanine7 azide (Cy7) was added and reacted overnight (8-12 hours) at 4°C. The resulting solution was placed on a 30% sucrose pad, centrifuged at 120,000 g and 4°C for 90 minutes, and the supernatant was removed to remove excess dye, yielding Cy3- and Cy7-labeled EVs (Cy3-EVs and Cy7-EVs), respectively.

[0062] 10 μM azide-labeled cNGR peptide was first reacted with DBCO-EVs at room temperature for 3 hours. Then, 10 μM Cyanine3azide (Cy3) or Cyanine7 azide (Cy7) was added, and the reaction was carried out overnight (8-12 hours) at 4°C. The resulting solution was placed on a 30% sucrose pad, centrifuged at 120,000 g at 4°C for 90 minutes, and the supernatant was removed to remove excess dye, yielding Cy3- and Cy7-labeled cNGR-EVs (Cy3-cNGR-EVs and Cy7-cNGR-EVs), respectively. Based on the fluorescence wavelength, Cy7-EVs and Cy7-cNGR-EVs were used in in vivo imaging experiments; Cy3-EVs and Cy3-cNGR-EVs were used in brain slice immunofluorescence experiments.

[0063] Nasal delivery: An anesthetic solution of ketamine:toluidine:PBS = 1:0.5:8.5 was prepared and administered to mice via a single intraperitoneal injection (ip), at a dose of 200 μL per 20 μg body weight. 10 μL of hyaluronidase (100 U) was administered bilaterally to the mice's nasal cavities to enhance nasal mucosal permeability. Using a 10 μL pipette, approximately 2 μL droplets were formed and brought close to the mouse's nostril, entering the nasal cavity with the inhalation airflow. This step was repeated at the other nostril of the same mouse after a 2-minute interval until the dose was completed, with permeability maintained for 30 minutes. After 30 minutes, the mice received 50 μL of PBS, 50 μL of 100 μg fluorescently labeled EVs, and cNGR-EVs via nasal administration, respectively.

[0064] In vivo tracking: Two hours after nasal delivery, mice were anesthetized (5% isoflurane induction, 2% isoflurane maintenance). After head preparation, near-infrared imaging of mice was performed using the VISQUEInVivo Smart-LF system, and the fluorescence radiation efficiency of regions of interest (ROIs) in the images was selected and analyzed. Surface functionalization does not affect the efficiency of EVs entering the brain via the nose. Figure 7 A).

[0065] Two hours after intranasal delivery, mice were anesthetized (ip, ketamine:toluidine:PBS = 1:0.5:8.5), and intracardiac perfusion was performed sequentially with pre-cooled PBS and 4% paraformaldehyde. After fixation in 4% paraformaldehyde and sucrose gradient dehydration, brain tissue was embedded in embedding medium and flash-frozen in liquid nitrogen. 30 μm brain slices were excised using a cryostat and incubated overnight at 4°C with PDGFR-β antibody, followed by incubation with the corresponding secondary antibody. Confocal microscopy was used to observe the colocalization of EVs and cNGR-EVs fluorescence with pericytes, and the percentage of pericytes taking up EVs or cNGR-EVs was counted. Surface functionalization significantly increased pericyte uptake of EVs (…). Figure 7 B).

[0066] Example 4

[0067] Treatment evaluation

[0068] Establishment of a bilateral common carotid artery stenosis (BCAS) model (i.e., vascular demyelinating model): A sham operation group (Sham) and groups receiving PBS, EVs, and cNGR-EVs via nasal delivery after BCAS were established. Mice were anesthetized (ip, ketamine:toluidine:PBS = 1:0.5:8.5), and anesthesia was maintained intraoperatively with 2% isoflurane. After exposing and disinfecting one side of the common carotid artery, a midline skin incision was made in the neck. Under a stereomicroscope, one side of the common carotid artery was exposed and dissected. Two silk sutures were brought out deep into the distal and proximal ends of the common carotid artery, respectively, and the artery was gently lifted and separated from the surrounding soft tissue using the sutures. A 0.18 mm inner diameter metal microcoil was wound around the common carotid artery to narrow it. After ensuring both ends of the coil were on the blood vessel, the silk sutures were removed. After a 30-minute interval, the other side of the common carotid artery was exposed and dissected, and another metal microcoil of the same size was wound around it. Carefully observe the anesthesia status of the mice during the operation. Before suturing, ensure there are no bleeding points and no obstruction of blood flow in the blood vessels. Disinfect the incision and suture. In the Sham group, only skin incision and carotid artery separation are performed, without micro-coil stenosis of the carotid artery. Place the mice on a warming pad after the operation and wait for recovery. After the mice recover, observe their activity and gait to ensure there is no hemiplegia or epileptic seizures caused by acute ischemic infarction. After the mice resume normal activity or foraging, anesthetize the mice (ip, ketamine: toluenethiazide: PBS = 1:0.5:8.5) and administer 10 μL of hyaluronidase (100 U) into both nasal cavities to enhance nasal mucosal permeability. 30 minutes later, the PBS, EVs, and cNGR-EVs groups received 50 μL of PBS and 50 μL of 100 μg of EVs or cNGR-EVs (prepared in Example 2) via the nose, respectively.

[0069] Laser speckle blood flow imaging for CBF detection: 30 days after BCAS surgery, mice were anesthetized (ip, ketamine:toluidine:PBS = 1:0.5:8.5) and fixed in a stereotactic frame. After scalp disinfection, a midline incision was made. The skull surface was cleaned with a cotton swab soaked in saline. Erythromycin ointment was used to protect the mice's eyes from laser stimulation. A thin film of saline was formed on the skull surface. The laser was turned on, and blood flow was recorded after the vascular morphology was fully visible on the screen and blood flow was basically stable. Compared with Sham, blood flow was significantly decreased in the PBS group; compared with the PBS and EVs groups, cerebral blood perfusion was significantly increased in the cNGR-EVs group. Figure 8 A).

[0070] Evans blue staining to detect BBB permeability: Three days after BCAS surgery, mice were anesthetized with 5% isoflurane and injected intraperitoneally with 2% Evans Blue. Two hours later, the brains were perfused and harvested. Direct observation revealed a significant blue exudate area in the cerebral cortex of mice in the PBS group compared to the Sham group; compared to the PBS and EVs groups, the blue exudate area in the cNGR-EVs group was reduced. Figure 8 B).

[0071] Electron microscopy examination of myelin pathology: Mice were anesthetized 30 days after BCAS (ip, ketamine:toluidine:PBS = 1:0.5:8.5) and perfused intracardiacly with 0.1 M PBS, followed by perfusion with a mixture of 4% paraformaldehyde and 1% glutaraldehyde (0.1 M phosphate buffer). Brain tissue was removed and fixed in 2.5% glutaraldehyde at 4 °C for 3 hours. 150 µm thick coronal sections were cut using a vibratory microtome, and callosity sections were prepared under a stereomicroscope. Electron microscopy revealed that compared with Sham, mice in the PBS group showed decreased axonal myelin density, structural abnormalities, and reduced myelin thickness, exhibiting demyelinating pathology; compared with the PBS and EVs groups, the cNGR-EVs group showed reduced demyelinating pathology (…). Figure 8 (C) This illustrates that the cNGR-EVs provided in the embodiments of the present invention can be used directly as drugs and have the potential to treat white matter damage caused by central nervous system diseases such as cerebral small vessel disease.

[0072] The cNGR-EVs formulation provided in the above embodiments of the present invention, administered via nasal administration, utilizes the specific binding of its targeting ligand cNGR to CD13 receptors on the surface of pericytes to precisely deliver therapeutic substances (such as neurotrophic factors, anti-inflammatory molecules, or nucleic acid drugs) to pericytes at the lesion site. It can also be directly used as a drug to treat CNS diseases. This application is particularly suitable for the early intervention stage of diseases, aiming to block or delay the pathological cascade triggered by pericyte dysfunction by protecting pericytes, salvaging cerebral blood flow, and strengthening the brain's blood flow barrier (BBB). This invention provides a novel targeted therapy strategy for central nervous system diseases such as cerebral small vessel disease, where pericyte dysfunction is an upstream event, for which effective early intervention methods are currently lacking.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An extracellular vesicle targeting brain pericytes, characterized in that, It includes extracellular vesicles and a targeting ligand modified on the surface of the extracellular vesicles, the targeting ligand specifically binding to cell surface receptors of pericytes.

2. The extracellular vesicles targeting brain pericytes according to claim 1, characterized in that, The targeting ligand is selected from the cyclic peptide cNGR; the cell surface receptor of the pericytes is selected from CD13.

3. The extracellular vesicles targeting brain pericytes according to claim 1, characterized in that, The targeting ligand is covalently coupled to the surface of the extracellular vesicles.

4. The extracellular vesicles targeting brain pericytes according to claim 3, characterized in that, The covalent reaction is selected from strain-promoted azide-alkyne cycloaddition reactions.

5. The extracellular vesicles targeting brain pericytes according to claim 4, characterized in that, The targeting ligand is coupled to the extracellular vesicle via the DBCO-NHS group.

6. The extracellular vesicles targeting brain pericytes according to any one of claims 1-5, characterized in that, The extracellular vesicles are loaded with therapeutic agents.

7. The use of the extracellular vesicles according to any one of claims 1-6 in the preparation of a medicament for treating diseases of the central nervous system.

8. The application according to claim 7, wherein the central nervous system disease is selected from cerebral small vessel disease.

9. A drug for treating diseases of the central nervous system, characterized in that, It includes the extracellular vesicles as described in any one of claims 1-6.

10. The medicament according to claim 8, characterized in that, The central nervous system diseases mentioned are selected from cerebral small vessel diseases.

Images